The overall objective of this competitive renewal application is to determine the molecular mechanisms responsible for Hyperhomocysteinemia (HHcy)-accelerated atherosclerosis. Major discoveries in the previous grant period include; 1) HHcy impairs endothelial function in severe HHcy CBS-/- mice by inhibiting eNOS expression and PKC activation, 2) HHcy inhibits post injury endothelial repair and lead to increase vascular remodeling in severe HHcy, 3) HHcy inhibits HDL biosynthesis via apo-AI inhibition in human and mouse CVD, 4) HHcy accelerates spontaneous atherosclerosis in CBS-/-/apoE-/- mice, 5) HHcy increased vessel wall content of cholesteryl ester (CE) and triglyceride (TG) contents and promoted MC uptake of Acetyl-LDL, 6) HHcy promoted inflammatory MC subset differentiation in hCBStg/mCBS-/-/apoE-/-. Collectively, these findings implicate HHcy in the etiology of inflammatory vascular diseases. The hypothesis to be tested in this proposal is that HHcy accelerates atherosclerosis by activating endothelium, promoting vessel wall inflammatory MC differentiation and increasing MC trans-endothelium migration. This project will study this hypothesis utilizing three linked specific aims. In Aim 1, we will examine the effects and mechanism of HHcy on endothelium activation and monocyte trans-endothelium migration using cultured primary endothelial and splenic cells in static condition or under physiological relevant flow. In Aim 2, we will study the role of HHcy on vessel wall MC origin and its relevance to atherogenesis using bone marrow transplantation from GFP mice into our newly developed HHcy mouse (CBS-/?/-). In Aim 3, we will study the effect of homocysteine-lowering on preventing MC trans-endothelium migration into the vessel wall and on reducing spontaneous atherosclerotic lesion formation. MC rolling/adhesion on EC will be examined in cremaster microcirculation model using intravital microscopy technology. We believe that completion of the specific Aims should Completion of the specific aims of this proposal may provide important insights into the role of Hcy in CVD, and identify the mechanistic links between HHcy and atherosclerosis. PUBLIC HEALTH RELEVANCE: Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular diseases (CVD). It has been suggested that HHcy accounts for the higher prevalence of CVD in renal disease, diabetes, ageing and in postmenopausal women that is not explained by traditional risk factors. However, the underlying mechanism is largely unknown and the role of homocysteine (Hcy)-induced vessel wall lipid accumulation has not been studies. We have demonstrated profound atherogenic effects of HHcy in disease mouse models established in our laboratory. We have recently reported that HHcy accelerates atherosclerosis, and increases inflammatory monocyte subsets in the peripheral tissues. In this proposal, we will use three connected Aims to examine the role and mechanisms of HHcy induced endothelial activation and monocyte-endothelium interaction. Aim 1 will examine the effects and mechanism of HHcy on endothelium activation and monocyte trans-endothelium migration using cultured primary endothelial and splenic cells in static condition or under physiological relevant flow. Aim 2 will study the role of HHcy on vessel wall MC origin and its relevance to atherogenesis using bone marrow transplantation from GFP mice into our newly developed HHcy mouse line (CBS/LRLR-/- mice). Aim 3 will study the effects of homocysteine-lowering on preventing MC trans-endothelium migration into the vessel wall and on reducing spontaneous atherosclerotic lesion formation.